Antimicrobial resistance has emerged as a health public concern as the combat of resistant microorganisms has become increasingly challenging. Thus, considering the urgent need of developing new therapeutic alternatives, the present study aimed to investigate the chemical composition and evaluate the antimicrobial and drug enhancing potential of the essential oil obtained from the leaves of Piper xylosteoides (Kunth) Steud (EOLPx) against clinically relevant bacterial and fungal strains. Following collection, the leaves were dried at room temperature in a dark environment and the extraction was performed by hydrodistillation using a modified Clevenger-type apparatus. The oil was dried with anhydrous magnesium sulfate, filtered, concentrated in rotary evaporator, and 5 mL aliquots were stored in a refrigerator. The oil yield was expressed as a percentage of the dry weight of the plant material (w/w). The chemical characterization was performed by gas chromatography coupled to mass spectrometry (GC–MS) and flame ionization detector (FID). The antimicrobial analysis was carried out using the broth microdilution and half-maximal inhibitory concentrations (IC50), minimum inhibitory concentrations (MIC), and minimum fungicidal concentrations (MFC) were deterimined by subculture. To evaluate the drug enhancing potential, subinhibitory concentrations (MFC/16 and MIC/8) of the essential oil were combined with the antifungal drug fluconazole and the antibiotics norfloxacin, gentamicin and erythromycin. The impact of the in vitro treatment on fungal virulence was determined by observing morphological transition in wet chambers. The chemical analysis identified the presence of monoterpenes (58.8%) and sesquiterpenes (35.3%), including the monoterpene hydrocarbon myrcene (27.5%) as the major component. The in vitro treatment with the essential oil caused little impact on C. albicans growth, showing a weak fungicidal effect against the URM 4387 strain (at 8192 µg/mL), while the INCQS 40006 strain was not affected. In addition, the association of the EOLPx with fluconazole did not enhance the antifungal effect of this drug against none of the C. albicans strains. However, both the natural product and fluconazole inhibited fungal pleomorphism by both strains. While the essential oil MIC against all bacterial strains was higher than 813 μg/mL, it was found to enhance the activity of gentamicin against the SA and EC isolates as well as to potentiate the activity of erythromycin against the EC isolate. On the other hand, antagonistic effects were observed from its association with norfloxacin for both strains. In conclusion, the EOLPx showed antibiotic-enhancing effects in addition to inhibiting C. albicans morphological transition. Nevertheless, further research is required to better characterize the antimicrobial effects and mechanisms of action by the essential oil components.